Human norovirus (NoV) causes an estimated 21 million cases of gastroenteritis in the United States each year (Scallan, et al., 2011). NoVs are members of the family Caliciviridae and contain a single stranded, positive sense RNA genome. NoVs are classified into five genogroups (GI-GV) and further subdivided into genotypes based on the capsid sequence (Zheng, et al., 2006; Kroneman, et al., 2011). Most human NoVs fall within the GI and GII genogroups. Norwalk virus (NV), genotype GI.1, was the first NoV isolated and is considered the prototype virus of the genus; nevertheless, genotype GII.4 NoVs are currently the most frequently detected in humans (Glass, et al., 2009). NoV infection is generally self-limiting in healthy adults, requiring only rehydration and supportive therapy; however, illness can be severe and even fatal in the elderly and young children (Glass, et al., 2009; Trivedi, et al., 2012). Furthermore, infection can be chronic in immunocompromised patients, lasting years and mimicking the symptoms of transplant rejection (Kaufmann, et al., 2005; Bok & Green, 2012).
NoV's low infectious dose, resistance to many common sterilization procedures, and ease of transmission make epidemic outbreaks common and difficult to control, particularly in semi-closed environments such as schools, nursing homes, hospitals, cruise ships, and military settings (Glass, et al., 2009; Hutson, et al., 2004). NoV has been shown to be the cause of the majority of nonbacterial gastroenteritis epidemics in the United States, resulting in a huge economic burden, particularly in healthcare settings, when considering the cost of lost revenue from closures to new admissions, staff absences, and cleaning expenses (Scallan, et al., 2011; Fankhauser, et al., 1998; Hoffmann, et al., 2012; Johnston, et al., 2007). The total annual cost of healthcare and lost productivity due to foodborne illness caused by norovirus in the USA is estimated to be $2 billion (Hoffman, et al., 2012).
In order to manage severe and chronic NoV infections and effectively control epidemic outbreaks, rapid diagnosis is crucial. In fact, it has been observed that outbreaks were contained an average of 6 days sooner (7.9 vs 15.4 days) when diagnosis was made within three days rather than four or more days after the first case, making a huge difference in the impact of the outbreak (Lopman, et al., 2004). Only one immunoassay, the RIDASCREEN Norovirus ELISA (3rd generation), is currently available for NoV diagnosis in the USA. However, this assay is currently approved for use only in the diagnosis of outbreaks because it lacks sensitivity. Moreover, negative results are unreliable as the sensitivity is highly dependent on the genotype associated with the outbreak (Parker, et al., 2005; Atmar, et al., 2001; Ambert-Balay & Pothier, 2012; Kirby, et al., 2010).
Phage display is a powerful technique for rapidly screening large-scale libraries for novel substrates (Sidhu, et al., 2003). Phage display employs random peptide sequences as fusions to geneIII protein, which is a solvent accessible capsid protein present in five copies on one end of the phage coat (Samoylova, et al., 2012). This allows biopanning in which the phage displaying peptide are allowed to interact with the chosen target, non-interacting phage are washed away, and specifically bound phage are eluted from the target by low pH. Several rounds of biopanning enrich for peptide sequences that interact with the target (Rakonjac, et al., 2011). The advantage of phage display relies on the presence of the genetic material for these peptides within the phage chromosome, linking the genotype and phenotype for easy determination of the sequence of the binding peptide (Paschke, 2003). This method has been used previously to isolate peptide-displaying phage that are useful diagnostic substrates for other bacterial and viral pathogens such as Mycobacterium avium and, more recently, hepatitis B virus (Monjezi, et al., 2012; Schofield, et al., 2012; Stratmann, et al., 2002).
The present disclosure provides a solution for a long-felt need in the art for detection of Norovirus. 